There is little doubt that cytochrome P450 CYP3A4 is the single most important protein in human xenobiotic metabolism. The prominence of CYP3A4 in drug metabolism results in routine investigation of the activity of thousands of molecules annually as substrates for this enzyme. Numerous computational methods have been developed to predict CYP3A4 metabolism. Ligand-based methods have considered structure-activity relationships for a baffling array of potential substrates, with a complex set of rather unreliable predictions. The key problem is that the conformational flexibility, or plasticity, of CYP3A4 has not been available and hence, has not been factored into these predictions. The long-term goal of this research program is to develop and apply novel approaches that dramatically expand our understanding of the P450 enzyme mechanisms. The objective of this application is to combine state-of-the-art tools of computational chemistry, chemical biology, and molecular spectroscopy to gain insight into the coupling of heme reactivity and protein dynamics, and the influence of interactions with redox partners with the adaptation of CYP3A4 to ligands. To meet this objective, there are three Specific Aims, we will: 1) Delineate the range of CYP3A4 conformational states in solution and the transition pathways between these conformers. Molecular dynamics methods capable of sampling low frequency protein motions will afford a description of CYP3A4 solution conformations, that to date, have been elusive by other means. 2) Define the functional consequences of ligand and redox partner interactions on CYP3A4 heme dynamics. Resonance Raman spectroscopy will be used to probe the conformational shifts and electronic structure changes resulting from interactions between CYP3A4, cytochrome P450 reductase, and cytochrome b5 that pre-organize that active site to facilitate electron transfer. 3) Map changes in CYP3A4 electrostatics by selective incorporation of Raman-active vibrational probes. The selective incorporation of amino acid analogs with vibrational probes will permit direct observation of local electrostatic changes through solvatochromic shifts induced by ligand binding, protein-protein interactions with redox partners, and resultant conformational interchanges. To afford accurate metabolic predictions for CYP3A4 metabolism, the conformational plasticity and the interactions between conformer and heme dynamics must be understood. We propose to approach these holes in our current understanding using a suite of interactive experimental designs. The significance of this set of studies is the promise of a clearer insight into ligand- and redox-partner induced changes in P450 dynamics and heme reactivity and the impact of these heretofore understudied factors in the adaptation of CYP3A4 to new substrate structures.